DOCSLIB.ORG

Somatic Hypermutation and Affinity Maturation Analysis Using the 4-Hydroxy-3-Nitrophenyl-Acetyl (NP) System

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Somatic Hypermutation and Affinity Maturation Analysis Using the 4-Hydroxy-3-Nitrophenyl-Acetyl (NP) System This is a repository copy of Somatic hypermutation and affinity maturation analysis using the 4-hydroxy-3-nitrophenyl-acetyl (NP) system. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/138157/ Version: Accepted Version Book Section: Heise, N and Klein, U orcid.org/0000-0002-4789-967X (2017) Somatic hypermutation and affinity maturation analysis using the 4-hydroxy-3-nitrophenyl-acetyl (NP) system. In: Calado, DP, (ed.) Germinal Centers. Methods in Molecular Biology, 1623 . Springer (Humana) , pp. 191-208. ISBN 978-1-4939-7094-0 https://doi.org/10.1007/978-1-4939-7095-7_16 (c) 2017, Springer Science+Business Media LLC. This is an author produced version of a chapter published in Methods in Molecular Biology volume 1623. Uploaded in accordance with the publisher's self-archiving policy. Reuse Items deposited in White Rose Research Online are protected by copyright, with all rights reserved unless indicated otherwise. They may be downloaded and/or printed for private study, or other acts as permitted by national copyright laws. The publisher or other rights holders may allow further reproduction and re-use of the full text version. This is indicated by the licence information on the White Rose Research Online record for the item. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Somatic hypermutation and affinity maturation analysis using the 4-hydroxy- 3-nitrophenyl-acetyl (NP) system Nicole Heise and Ulf Klein Herbert Irving Comprehensive Cancer Center and Departments of Pathology & Cell Biology and Microbiology & Immunology, Columbia University, 1130 St. Nicholas Avenue R312, New York, NY 10032, USA Correspondence to U.K. email: [email protected] Running head: NP-system in analysis of affinity maturation 1 Abstract Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell- dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino-acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to i) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to ii) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response. Key Words B cell; plasma cell; Ig variable region gene; germinal center; somatic hypermutation; affinity maturation; T cell-dependent; T cell-independent 2 1. Introduction Antigen-specific memory B cells and plasma cells are generated during the germinal center (GC) reaction of T cell-dependent immune responses in secondary lymphoid tissues (1, 2). Within the GC microenvironment, antigen-activated B cells undergo somatic hypermutation of the rearranged IgV genes with the aim to generate high-affinity antibodies that effectively bind to the invading pathogen, resulting in its elimination. In a defined area within the GC called dark zone, the rapidly proliferating ‘dark zone’ cells hypermutate their IgV genes and then differentiate into ‘light zone’ cells that are positively selected for improved antigen-binding in the GC light zone (2-5). Selected light zone B cells recirculate to the dark zone to undergo additional rounds of mutation and selection to further improve antigen-affinity before they eventually differentiate into memory B cells or plasma cells that exit the GC. The descendants of the GC reaction thus carry somatically mutated IgV genes and have often switched from IgM to other Ig classes which have different effector functions. Understanding the molecular mechanism of the GC response is critical for the development of improved vaccines against microorganisms. Thus, for studies aimed at manipulating the GC response, which e.g. may include the deletion or overexpression of GC- associated genes or the use of particular adjuvants, it is imperative to have tools that allow the investigation of the processes that underlie the generation of antigen-specific memory B cells and plasma cells, i.e. somatic hypermutation and antibody affinity maturation. In C57/Bl6 mice, the immune response against the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein frequently results in a specific, affinity-enhancing hypermutation in codon 33 of the V186.2 gene segment that leads to an amino acid exchange resulting in a ~10-fold increase in affinity against NP (6-8), allowing for the generation of NP-specific GC B cells, memory B cells 3 and plasma cells to be tracked at the molecular level. The resulting antibodies are mostly of the IgG1 class (6-8). The characterization of the B-cell response against NP at the molecular level by several groups (9-11) provided the baseline for numerous studies on the extent of affinity maturation in situations where certain genes were either knocked out in the mouse germ-line or conditionally deleted in GC B cells (12-16). By immunizing mice with NP coupled to a carrier protein such as chicken gammaglobulin (CGG) or keyhole limpet hemocyanin (KLH), the characteristics and dynamics of the immune response against the hapten in the presence or absence of a certain signaling pathway or transcription factor, or a specific treatment, can be investigated by i) determining the somatic hypermutation frequency in V186.2 gene rearrangements, ii) the fraction of B cells with the affinity-enhancing tryptophan to leucine amino-acid exchange, iii) the quantity of NP- specific serum IgG1 secreted by GC-derived plasma cells by ELISA, and iv) the frequency of plasma cells in lymphoid tissues by ELISPOT analysis. The use of the NP system is not confined to T cell-dependent antibody responses, as the coupling of NP to lipopolysaccharide (LPS) or the polysaccharide Ficoll allows the study of the plasma cell response in T cell-independent responses type I and II, respectively, by ELISA. 2. Materials 2.1 Immunization Sterile phosphate-buffered saline (PBS). NP-KLH (e.g. NP28-KLH, Biosearch Technologies; of note, the number of NP molecules conjugated to KLH varies among batches). 4 For T-independent immunizations (Notes 1), NP-LPS and NP-AECM-FICOLL (Biosearch Technologies). Freud's adjuvant complete. Freud's adjuvant incomplete. 1 ml syringes. 5 Needles (18G1½ and 25G /8). Vortex. Sonicator. 2.2 Acquisition of blood samples and serum preparation Razor blade or scalpel. 1 ml or 2 ml syringes. 21G1 needles. 1.5 ml collection tubes. Centrifuge. 2.3 ELISA 96-well immune plates (Thermo Fisher Scientific). 96-well culture plates. Parafilm. PBS. 2% fetal bovine serum (FBS) in PBS. Wash buffer: PBS containing 0.05% Tween-20. Capture antibody: anti-mouse Ig(H+L) (Southern Biotec). 5 NP-bovine serum albumin (BSA) with low and high hapten coating (e.g. NP9-BSA and NP25-BSA). Mouse Ig (e.g. IgM, IgG1). Detection antibody: alkaline phosphatase (AP)-conjugated anti-mouse Ig (e.g. IgM, IgG1). PNPP (p-nitrophenylphosphate; Southern Biotec). Substrate buffer (500 ml): ddH2O + 24.5 mg MgCl2x6H2O + 48 ml diethanolamine; adjust pH to 9.8 with 5N HCl. Microplate reader. 2.4 Sample generation from mouse tissues following NP immunization PBS/0.5% BSA Glass slides; alternatively 40 µm cell strainer, 50 ml falcon tube and plunger of a 5 ml syringe. 10 ml syringes. 22G1 needles. RBC lysis buffer (e.g. Red Blood Cell Lysis HybriMax; Sigma Aldrich); alternatively, prepare lysis buffer: ammonium chloride (NH4Cl) 0.15 M, 8.29 g per l; potassium bicarbonate (KHCO3) 10 mM, 1 g per l; EDTA 0.1 mM, 0.037 g per l; H2O, 1 l; filter solution with 0.45 m filter, store at 4ºC. 15 ml Falcon tubes. Refrigerated centrifuge. Optional: cell counter (e.g. Countess, Invitrogen). 6 2.5 ELISPOT 96-well filtration plates (Millipore, Cat# MSIPS4510). 96-well culture plates. Plastic wrap. 0.2 µm filter. 35% ethanol (diluted in molecular grade H2O). Sterile PBS. RPMI culture medium supplemented with 10% fetal bovine serum (FBS), 1% Penicillin/Streptomycin and 0.1% -mercaptoethanol. Wash buffer: PBS containing 0.05% Tween-20. PBS/2% BSA. NP-BSA with high hapten coating (e.g. NP25-BSA). Detection antibody: AP-conjugated anti-mouse IgG1. Nitro blue tetrazolium chloride-5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP; Roche). NBT/BCIP substrate buffer: 0.1 M Tris + 0.1 M NaCl + 0.05 M MgCl2; pH 9.5. Optional: cell counter. Microplate reader (alternatively, colonies can be counted using a magnifying glass and pictures of wells can be taken with a camera that has a macro function). 2.6 Isolation of B-cell subsets from immunized mice by FACS RBC lysis buffer (see above). PBS/0.5% BSA.
Load more

Recommended publications
  • B Cell Activation and Escape of Tolerance Checkpoints: Recent Insights from Studying Autoreactive B Cells
    cells Review B Cell Activation and Escape of Tolerance Checkpoints: Recent Insights from Studying Autoreactive B Cells Carlo G. Bonasia 1 , Wayel H. Abdulahad 1,2 , Abraham Rutgers 1, Peter Heeringa 2 and Nicolaas A. Bos 1,* 1 Department of Rheumatology and Clinical Immunology, University Medical Center Groningen, University of Groningen, 9713 Groningen, GZ, The Netherlands; [email protected] (C.G.B.); [email protected] (W.H.A.); [email protected] (A.R.) 2 Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, 9713 Groningen, GZ, The Netherlands; [email protected] * Correspondence: [email protected] Abstract: Autoreactive B cells are key drivers of pathogenic processes in autoimmune diseases by the production of autoantibodies, secretion of cytokines, and presentation of autoantigens to T cells. However, the mechanisms that underlie the development of autoreactive B cells are not well understood. Here, we review recent studies leveraging novel techniques to identify and characterize (auto)antigen-specific B cells. The insights gained from such studies pertaining to the mechanisms involved in the escape of tolerance checkpoints and the activation of autoreactive B cells are discussed. Citation: Bonasia, C.G.; Abdulahad, W.H.; Rutgers, A.; Heeringa, P.; Bos, In addition, we briefly highlight potential therapeutic strategies to target and eliminate autoreactive N.A. B Cell Activation and Escape of B cells in autoimmune diseases. Tolerance Checkpoints: Recent Insights from Studying Autoreactive Keywords: autoimmune diseases; B cells; autoreactive B cells; tolerance B Cells. Cells 2021, 10, 1190. https:// doi.org/10.3390/cells10051190 Academic Editor: Juan Pablo de 1.
    [Show full text]
  • Case of the Anti HIV-1 Antibody, B12
    Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: Roland Strong, Chair Nancy Maizels Jessica A. Hamerman Program Authorized to Offer Degree: Molecular and Cellular Biology i ii ©Copyright 2015 Mesfin Mulugeta Gewe iii University of Washington Abstract Spectrum of Somatic Hypermutations and Implication on Antibody Function: Case of the anti HIV-1 antibody, b12 Mesfin Mulugeta Gewe Chair of the Supervisory Committee: Roland Strong, Full Member Fred Hutchinson Cancer Research Center Sequence diversity, ability to evade immune detection and establishment of human immunodeficiency virus type 1 (HIV-1) latent reservoirs present a formidable challenge to the development of an HIV-1 vaccine. Structure based vaccine design stenciled on infection elicited broadly neutralizing antibodies (bNAbs) is a promising approach, in some measure to circumvent existing challenges. Understanding the antibody maturation process and importance of the high frequency mutations observed in anti-HIV-1 broadly neutralizing antibodies are imperative to the success of structure based vaccine immunogen design. Here we report a biochemical and structural characterization for the affinity maturation of infection elicited neutralizing antibodies IgG1 b12 (b12). We investigated the importance of affinity maturation and mutations accumulated therein in overall antibody function and their potential implications to vaccine development. Using a panel of point iv reversions, we examined relevance of individual amino acid mutations acquired during the affinity maturation process to deduce the role of somatic hypermutation in antibody function.
    [Show full text]
  • Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for A
    International Journal of Molecular Sciences Article Affinity Maturation of a T-Cell Receptor-Like Antibody Specific for a Cytomegalovirus pp65-Derived Peptide Presented by HLA-A*02:01 Se-Young Lee 1,†, Deok-Han Ko 1,†, Min-Jeong Son 1, Jeong-Ah Kim 1, Keunok Jung 2 and Yong-Sung Kim 1,2,* 1 Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea; [email protected] (S.-Y.L.); [email protected] (D.-H.K.); [email protected] (M.-J.S.); [email protected] (J.-A.K.) 2 Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon 16499, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-31-219-2662; Fax: +82-31-219-1610 † These authors have contributed equally to this work. Abstract: Human cytomegalovirus (CMV) infection is widespread among adults (60–90%) and is usually undetected in healthy individuals without symptoms but can cause severe diseases in im- munocompromised hosts. T-cell receptor (TCR)-like antibodies (Abs), which recognize complex antigens (peptide–MHC complex, pMHC) composed of MHC molecules with embedded short pep- tides derived from intracellular proteins, including pathogenic viral proteins, can serve as diagnostic and/or therapeutic agents. In this study, we aimed to engineer a TCR-like Ab specific for pMHC comprising a CMV pp65 protein-derived peptide (495NLVPMVATV503; hereafter, CMVpp65 ) 495-503 in complex with MHC-I molecule human leukocyte antigen (HLA)-A*02:01 (CMVpp65495-503/HLA- A*02:01) to increase affinity by sequential mutagenesis of complementarity-determining regions Citation: Lee, S.-Y.; Ko, D.-H.; Son, M.-J.; Kim, J.-A.; Jung, K.; Kim, Y.-S.
    [Show full text]
  • What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination?: a Role for Antibody Feedback
    Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press What Are the Primary Limitations in B-Cell Affinity Maturation, and How Much Affinity Maturation Can We Drive with Vaccination? A Role for Antibody Feedback Kai-Michael Toellner,1 Daniel M.-Y. Sze,2 and Yang Zhang1 1Institute of Immunology and Immunotherapy, University of Birmingham, Birmingham B15 2TT, United Kingdom 2School of Health and Biomedical Sciences, RMIT University, Bundoora 3083, Australia Correspondence: [email protected] We discuss the impact of antibody feedback on affinity maturation of B cells. Competition from epitope-specific antibodies produced earlier during the immune response leads to immune complex formation, which is essential for transport and deposition of antigen onto follicular dendritic cells (FDCs). It also reduces the concentration of free epitopes into the mM to nM range, which is essential for B-cell receptors (BCRs) to sense affinity-dependent changes in binding capacity. Antibody feedback may also induce epitope spreading, leading to a broader selection of epitopes recognized by newly emerging B-cell clones. This may be exploitable, providing ways to manipulate epitope usage induced by vaccination. GREAT DEBATES What are the most interesting topics likely to come up over dinner or drinks with your colleagues? Or, more importantly, what are the topics that don’t come up because they are a little too controversial? In Immune Memory and Vaccines: Great Debates, Editors Rafi Ahmed and Shane Crotty have put together a collection of articles on such ques- tions, written by thought leaders in these fields, with the freedom to talk about the issues as they see fit.
    [Show full text]
  • Vaccine Immunology Claire-Anne Siegrist
    2 Vaccine Immunology Claire-Anne Siegrist To generate vaccine-mediated protection is a complex chal- non–antigen-specifc responses possibly leading to allergy, lenge. Currently available vaccines have largely been devel- autoimmunity, or even premature death—are being raised. oped empirically, with little or no understanding of how they Certain “off-targets effects” of vaccines have also been recog- activate the immune system. Their early protective effcacy is nized and call for studies to quantify their impact and identify primarily conferred by the induction of antigen-specifc anti- the mechanisms at play. The objective of this chapter is to bodies (Box 2.1). However, there is more to antibody- extract from the complex and rapidly evolving feld of immu- mediated protection than the peak of vaccine-induced nology the main concepts that are useful to better address antibody titers. The quality of such antibodies (e.g., their these important questions. avidity, specifcity, or neutralizing capacity) has been identi- fed as a determining factor in effcacy. Long-term protection HOW DO VACCINES MEDIATE PROTECTION? requires the persistence of vaccine antibodies above protective thresholds and/or the maintenance of immune memory cells Vaccines protect by inducing effector mechanisms (cells or capable of rapid and effective reactivation with subsequent molecules) capable of rapidly controlling replicating patho- microbial exposure. The determinants of immune memory gens or inactivating their toxic components. Vaccine-induced induction, as well as the relative contribution of persisting immune effectors (Table 2.1) are essentially antibodies— antibodies and of immune memory to protection against spe- produced by B lymphocytes—capable of binding specifcally cifc diseases, are essential parameters of long-term vaccine to a toxin or a pathogen.2 Other potential effectors are cyto- effcacy.
    [Show full text]
  • B-Cell Development, Activation, and Differentiation
    B-Cell Development, Activation, and Differentiation Sarah Holstein, MD, PhD Nov 13, 2014 Lymphoid tissues • Primary – Bone marrow – Thymus • Secondary – Lymph nodes – Spleen – Tonsils – Lymphoid tissue within GI and respiratory tracts Overview of B cell development • B cells are generated in the bone marrow • Takes 1-2 weeks to develop from hematopoietic stem cells to mature B cells • Sequence of expression of cell surface receptor and adhesion molecules which allows for differentiation of B cells, proliferation at various stages, and movement within the bone marrow microenvironment • Immature B cell leaves the bone marrow and undergoes further differentiation • Immune system must create a repertoire of receptors capable of recognizing a large array of antigens while at the same time eliminating self-reactive B cells Overview of B cell development • Early B cell development constitutes the steps that lead to B cell commitment and expression of surface immunoglobulin, production of mature B cells • Mature B cells leave the bone marrow and migrate to secondary lymphoid tissues • B cells then interact with exogenous antigen and/or T helper cells = antigen- dependent phase Overview of B cells Hematopoiesis • Hematopoietic stem cells (HSCs) source of all blood cells • Blood-forming cells first found in the yolk sac (primarily primitive rbc production) • HSCs arise in distal aorta ~3-4 weeks • HSCs migrate to the liver (primary site of hematopoiesis after 6 wks gestation) • Bone marrow hematopoiesis starts ~5 months of gestation Role of bone
    [Show full text]
  • B Cell Responses Against Influenza Viruses
    viruses Review B Cell Responses against Influenza Viruses: Short-Lived Humoral Immunity against a Life-Long Threat Jenna J. Guthmiller 1,* , Henry A. Utset 1 and Patrick C. Wilson 1,2 1 Section of Rheumatology, Department of Medicine, University of Chicago, Chicago, IL 60637, USA; [email protected] (H.A.U.); [email protected] (P.C.W.) 2 Committee on Immunology, University of Chicago, Chicago, IL 60637, USA * Correspondence: [email protected] Abstract: Antibodies are critical for providing protection against influenza virus infections. However, protective humoral immunity against influenza viruses is limited by the antigenic drift and shift of the major surface glycoproteins, hemagglutinin and neuraminidase. Importantly, people are exposed to influenza viruses throughout their life and tend to reuse memory B cells from prior exposure to generate antibodies against new variants. Despite this, people tend to recall memory B cells against constantly evolving variable epitopes or non-protective antigens, as opposed to recalling them against broadly neutralizing epitopes of hemagglutinin. In this review, we discuss the factors that impact the generation and recall of memory B cells against distinct viral antigens, as well as the immunological limitations preventing broadly neutralizing antibody responses. Lastly, we discuss how next-generation vaccine platforms can potentially overcome these obstacles to generate robust and long-lived protection against influenza A viruses. Keywords: influenza viruses; humoral immunity; broadly neutralizing antibodies; imprinting; Citation: Guthmiller, J.J.; Utset, H.A.; hemagglutinin; germinal center; plasma cells Wilson, P.C. B Cell Responses against Influenza Viruses: Short-Lived Humoral Immunity against a Life-Long Threat. Viruses 2021, 13, 1.
    [Show full text]
  • The Regulation of Peptide Mimotope/Epitope Recognition By
    The Regulation of Peptide Mimotope/Epitope Recognition by Monoclonal Antibodies by Richard Gary Smith B.Sc. (Hons) Thesis submitted to the University of Nottingham for the degree of Doctor of Philosophy, March 2002. TABLE OF CONTENTS Abstract………………………………………………………………...……….…….8 Acknowledgements…………………………………………………………………...9 Glossary of terms……………………………………………………………………10 Chapter 1 Antibody Purification…………………………………………………..11 1.1 Introduction ......................................................................................................11 1.1.1 Antibodies ................................................................................................11 1.1.2 Monoclonal and engineered Antibodies...................................................12 1.2 Examples of a therapeutic and a commercially applied monoclonal antibody17 1.2.1 C595 monoclonal antibody (NCRC48)...................................................17 1.2.2 mAb 4155.................................................................................................18 1.3 Approaches to antibody purification................................................................20 1.3.1 Column chromatography..........................................................................20 1.3.2 Fluidized bed and expanded bed adsorption ............................................20 1.3.3 High performance Tangential Flow Filtration (HPTFF)..........................21 1.3.4 Magnetic beads.........................................................................................21 1.4 Physical
    [Show full text]
  • In Vitro Selection and Affinity Maturation of Antibodies from A
    Proc. Natd. Acad. Sci. USA Vol. 89, pp. 3576-3580, April 1992 Biochemistry In vitro selection and affinity maturation of antibodies from a naive combinatorial immunoglobulin library (naive immunoglobulin repertoire/fflamentous phage/random mutagenesis) HERMANN GRAM*, LORI-ANNE MARCONI, CARLOS F. BARBAS III, THOMAS A. COLLET, RICHARD A. LERNER, AND ANGRAY S. KANG The Scripps Research Institute, Departments of Molecular Biology and Chemistry, 10666 North Torrey Pines Road, La Jolla, CA 92037 Contributed by Richard A. Lerner, January 7, 1992 ABSTRACT We have used a combinatorial immunoglob- H-chain IgG isotype for molecular cloning, and/or the high ulin library approach to obtain monoclonal antibodies from abundance of specific mRNA in the starting material (12) nonimmune adult mice, thereby establishing the principles of most likely biased the combinatorial libraries toward affinity- (i) accessing naive combinatorial antibody libraries for prede- matured antibodies. Recently, Marks et al. (19) demonstrated termined specificities and (ii) increasing the affinity of the that active single-chain (Sc) antibodies with affinity constants selected antibody binding sites by random mutagenesis. A between 106 and 107 M-1 against a hapten or a small number combinatorial Fab library expressing immunoglobulin Ip and it of epitopes on a protein can be obtained directly from light-chain fragments on the surface of filamentous phage was nonimmune combinatorial immunoglobulin libraries. prepared from bone marrow ofnonimmunized, adult BALB/c In its simplest form, the initial stage of the immune mice with the multivalent display vector pComb8. Phage response can be re-created in vitro by generating a combi- displaying low affinity Fabs (binding constants, 104-1O M-') natorial library of PCR-amplified immunoglobulin At and L binding to a progesterone-bovine serum albumin conjugate chains from the bone marrow of adult mice.
    [Show full text]
  • Generation of Memory B Cells Inside And
    1258 Toshitada Takemori et al. DOI: 10.1002/eji.201343716 Eur. J. Immunol. 2014. 44: 1258–1264 Mini-Review Generation of memory B cells inside and outside germinal centers Toshitada Takemori1, Tomohiro Kaji2, Yoshimasa Takahashi3, Michiko Shimoda4 and Klaus Rajewsky5 1 Drug Discovery Antibody Platform Unit, RIKEN Center for Integrative Medical Sciences (IMS), Yokohama, Japan 2 Laboratory for Immunological Memory, RIKEN Research Center for Allergy and Immunology (RCAI), Yokohama, Japan 3 Department of Immunology, National Institute of Infectious Diseases, Tokyo, Japan 4 Cancer Immunology, Inflammation and Tolerance Program, Georgia Regents University Cancer Center, Augusta, GA, USA 5 Immune Regulation and Cancer, Max-Delbruck-Center¨ for Molecular Medicine, Berlin, Germany Germinal centers (GCs) are generally considered to be the sole site of memory B-cell generation. However, recent studies demonstrate that memory B cells can also develop in response to a T-cell dependent (TD) antigen before the onset, and independently of, the GC reaction. These two classes of memory cells persist equally over long periods of time and attain functional maturation through distinct but related transcriptional programs. Although the development of both memory B-cell types requires classical T-cell help, the generation of GC-dependent memory B cells requires TFH-cell help, while the generation of GC-independent memory cells does not. These findings led to the conclusion that B-cell memory is generated along two fundamentally distinct cellular differentiation pathways. In this review, we focus on the GC-independent pathway of memory B-cell development, and discuss how the unique features of memory B cells are maintained in the GC-independent pathway.
    [Show full text]
  • γδ T Cells Control Humoral Immune Response by Inducing T Follicular
    ARTICLE DOI: 10.1038/s41467-018-05487-9 OPEN γδ T cells control humoral immune response by inducing T follicular helper cell differentiation Rafael M. Rezende1, Amanda J. Lanser1, Stephen Rubino1, Chantal Kuhn1, Nathaniel Skillin1, Thais G. Moreira1, Shirong Liu1, Galina Gabriely1, Bruna A. David2, Gustavo B. Menezes2 & Howard L. Weiner1 γδ T cells have many known functions, including the regulation of antibody responses. However, how γδ T cells control humoral immunity remains elusive. Here we show that 1234567890():,; complete Freund’s adjuvant (CFA), but not alum, immunization induces a subpopulation of CXCR5-expressing γδ T cells in the draining lymph nodes. TCRγδ+CXCR5+ cells present antigens to, and induce CXCR5 on, CD4 T cells by releasing Wnt ligands to initiate the T follicular helper (Tfh) cell program. Accordingly, TCRδ−/− mice have impaired germinal center formation, inefficient Tfh cell differentiation, and reduced serum levels of chicken ovalbumin (OVA)-specific antibodies after CFA/OVA immunization. In a mouse model of lupus, TCRδ−/− mice develop milder glomerulonephritis, consistent with decreased serum levels of lupus-related autoantibodies, when compared with wild type mice. Thus, modulation of the γδ T cell-dependent humoral immune response may provide a novel therapy approach for the treatment of antibody-mediated autoimmunity. 1 Ann Romney Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA. 2 Center for Gastrointestinal Biology, Federal University of Minas Gerais,
    [Show full text]
  • Self-Antigen Driven Affinity Maturation Is Required for Pathogenic
    bioRxiv preprint doi: https://doi.org/10.1101/2020.03.14.988758; this version posted March 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Self-antigen driven affinity maturation is required for pathogenic monovalent IgG4 autoantibody development Miriam L. Fichtner1,2,*, Casey Vieni3,4,*, Rachel L. Redler3, Ljuvica Kolich3, Ruoyi Jiang2, Kazushiro Takata1,2, Panos Stathopoulos1,2, Pablo Suarez1,2, Richard J. Nowak1, Steven J. Burden3, Damian C. Ekiert3,§ and Kevin C. O’Connor1,2,§. 1. Department of Neurology, Yale University School of Medicine, New Haven, CT 06511, USA 2. Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06511, USA 3. Departments of Cell Biology and Microbiology, New York University School of Medicine, New York, NY 10016, USA 4. Medical Scientist Training Program, New York University School of Medicine, New York, NY, USA *, § Authorship note: First authors MLF and CV, and senior authors DCE and KCO contributed equally to this work. Corresponding author and contact information: Kevin C. O’Connor, PhD Yale School of Medicine Departments of Neurology and Immunobiology 300 George Street - Room 353J New Haven, CT 06511 Phone: (203)-737-3321 Fax: (203)-737-7903 [email protected] bioRxiv preprint doi: https://doi.org/10.1101/2020.03.14.988758; this version posted March 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Summary IgG4 autoantibodies in autoimmune myasthenia gravis are functionally monovalent, requiring a high affinity threshold featuring fast on/slow off rates, to reach pathogenic capacity.
    [Show full text]